Establishing the Thermodynamic Cards of Dipine Models’ Oxidative Metabolism on 21 Potential Elementary Steps

Dipines are a type of important antihypertensive drug as L-calcium channel blockers, whose core skeleton is the 1,4-dihydropyridine structure. Since the dihydropyridine ring is a key structural factor for biological activity, the thermodynamics of the aromatization dihydropyridine ring is a significant feature parameter for understanding the mechanism and pathways of dipine metabolism in vivo. Herein, 4-substituted-phenyl-2,6-dimethyl-3,5-diethyl-formate-1,4-dihydropyridines are refined as the structurally closest dipine models to investigate the thermodynamic potential of dipine oxidative metabolism. In this work, the thermodynamic cards of dipine models’ aromatization on 21 potential elementary steps in acetonitrile have been established. Based on the thermodynamic cards, the thermodynamic properties of dipine models and related intermediates acting as electrons, hydrides, hydrogen atoms, protons, and two hydrogen ions (atoms) donors are discussed. Moreover, the thermodynamic cards are applied to evaluate the redox properties, and judge or reveal the possible oxidative mechanism of dipine models.

Our group has long been committed to thermodynamic research on hydrogen transfer for NADH models, and determining the thermodynamic driving forces of over 200 ΔG HD (DH 2 ) ΔG HD (DH 2 ) ΔG PD DH' ) Step 1 Step 4 Step 2 Step 5 Step 3 Step 13 Step 20 Step 15 Step 8 Step 10 Step 9 Step 11 Step 7 Step 12 Step 6 S t e p 1 8 S t e p 1 7
Our group has long been committed to thermodynamic research on hydrogen transfer for NADH models, and determining the thermodynamic driving forces of over 200 organic hydrides releasing hydrides in non-aqueous media [27,31].The previous works inspire us to further investigate and clarify the thermodynamic parameters of dipine aromatization in solution.Herein, the thermodynamic cards of dipine models' aromatization on 21 potential elementary steps has been established (Figure 3).From the thermodynamic cards, the thermodynamic properties of dipine models and related intermediates acting as electrons, hydrides, hydrogen atoms, protons, and two hydrogen ions (atoms) donors are discussed.Furthermore, the thermodynamic cards are utilized to measure the redox properties, and diagnose the possible oxidative mechanism of dipine models.
Steps 2-3 and Steps 8-9 are the electron-releasing chemical processes, and their thermodynamic potentials are described by the related oxidation potentials, E ox (DH 2 ) for Step 2, E ox (DH • ) for Step 3, E ox (DH − ) for Step 8, and E ox (D •− ) for Step 9, respectively.The oxidation potentials were determined in our previous work [27,38].
Step 19 is the chemical process of DH 2 releasing hydrogen gas (H 2 ), and the thermodynamic potential is described by the Gibbs free energy of DH 2 releasing H 2 , ∆G H2 (DH 2 ).∆G H2 (DH 2 ) could be obtained by Equation (15) in Table 1, 15)).Among Equation (15), ∆G H − D (H 2 ) refers to the Gibbs free energy of H 2 releasing hydrides, which was reported as 76.0 kcal/mol [41,42] in acetonitrile.
Step 20 is the chemical process of a hydrogen atom transfer within a molecule from the N 1 -position to the C 4 -position, and the thermodynamic potential is described by the related Gibbs free energy, ∆G HT (DH • ).∆G HT (DH • ) could be obtained by Equation (16) in Table 1, ∆G HT (DH • ) = ∆G HD (DH • ) − ∆G HD (DH' • ) (Equation ( 16)) by constructing the thermodynamic cycle [27] Step 20-Step 11-Step 17.
Table 1.Chemical processes, thermodynamic potentials, and computed equations or data sources of 21 elementary steps for DH 2 aromatization.
All of the chemical processes, thermodynamic potentials, computed equations, or data sources of the 21 elementary steps for DH 2 aromatization are listed in Table 1.Moreover, the thermodynamic results of 1H 2 -5H 2 and YH 2 aromatization are shown in Table 2. Accordingly, the thermodynamic cards [27,31,38] of 1H 2 -5H 2 (Figures S1-S5) and YH 2 (Figure S6) on 21 potential elementary steps are established and presented in the Supporting Information to make them more convenient to query and use.Obviously, the thermodynamic cards (Figures 3 and S1-S5) can visually exhibit the mutual transformations among dipine models DH 2 , aromatic products D, and the related six active intermediates, as well as the thermodynamic driving forces of the related 21 potential elementary steps.Naturally, the thermodynamic cards could be employed to quantitatively measure and predict the characteristic chemical or thermodynamic properties of the dipine models and involved intermediates.

Thermodynamic Properties of DH 2 and Related
Intermediates Acting as Electrons, Hydrides, Hydrogen Atoms, Protons, and Two Hydrogen Ions (Atoms) Donors in Acetonitrile 2.

Hydride-Donating Properties
For a better comparison, the hydride-donating properties of DH 2 and DH − and the hydricities of DH 2 , DH − , and common hydride donors (iAscH − , BNAH, and AcrH), as well as the hydride-affinities of common coenzyme models (BNA + for NADH coenzyme, PQ for coenzyme Q, iAsc for oxidated ascorbic acid, Fl + for flavin coenzyme, and Ru IV O 2+ for heme enzyme) and hydride acceptors (H + and PTZ •+ ) in acetonitrile are displayed in Figure 6.As can be seen from Figure 6, it is found that the hydricity ranges from 63.9 to 69.0 kcal/mol for DH 2 , and from 31.8 to 37.9 kcal/mol for DH − .If the hydricities between DH 2 and DH − are compared, it is easy to discover that the hydricities of DH − are greatly improved by the negative charge at the N 1 -atom compared with their parents DH 2 , and the hydride-donating abilities of DH − (31.8-37.9kcal/mol) are greater than DH 2 (63.9-69.0kcal/mol) by more than 30 kcal/mol.Based on their thermodynamic range, DH 2 (63.9-69.0kcal/mol) belong to thermodynamically medium-strong hydride donors, while DH − (31.8-37.9kcal/mol) belong to thermodynamically strong hydride donors, respectively.As can be seen from Figure 6, it is found that the hydricity ranges from 63.9 to 69.0 kcal/mol for DH2, and from 31.8 to 37.9 kcal/mol for DH − .If the hydricities between DH2 and DH − are compared, it is easy to discover that the hydricities of DH − are greatly improved by the negative charge at the N1-atom compared with their parents DH2, and the hydride-donating abilities of DH − (31.8-37.9kcal/mol) are greater than DH2 (63.9-69.0kcal/mol) by more than 30 kcal/mol.Based on their thermodynamic range, DH2 (63.9-69.0kcal/mol) belong to thermodynamically medium-strong hydride donors, while DH − (31.8-37.9kcal/mol) belong to thermodynamically strong hydride donors, respectively.DH2 are thermodynamically worse hydride donors than BNAH and thermodynamically better hydride donors than iAscH − , due to the hydricities of DH2 (63.9-69.0kcal/mol) being more negative than BNAH (59.3 kcal/mol) [27]   DH 2 are thermodynamically worse hydride donors than BNAH and thermodynamically better hydride donors than iAscH − , due to the hydricities of DH 2 (63.9-69.0kcal/mol) being more negative than BNAH (59.3 kcal/mol) [27] by 4.6-9.7 kcal/mol, and greater than iAscH − (75.7 kcal/mol) [27] by 6.7-11.8kcal/mol.DH − are much better hydride donors than BNAH and iAscH − , and the hydricities of DH − (31.8-37.9kcal/mol) are much greater than BNAH (59.3 kcal/mol) and iAscH − (75.7 kcal/mol) by more than 20 kcal/mol.The above thermodynamic data indicate that DH 2 could not be oxidated by BNA + through hydride transfer with related Gibbs free energies greater than 0, 4.6 kcal/mol ≤ ∆G H -T (DH 2 /BNA + ) ≤ 9.7 kcal/mol, but the anion intermediates of DH 2 (DH − ) could be oxidated by BNA + through hydride transfer with Gibbs free energies less than 0, −27.5 kcal/mol ≤ ∆G H -T (DH − /BNA + ) ≤ −21.4 kcal/mol.In addition, DH 2 and DH − could be oxidated by iAsc (−75.7 kcal/mol) by hydride transfer with Gibbs free energies less than 0, −9.7 kcal/mol ≤ ∆G H -T (DH 2 /iAsc) ≤ −4.6 kcal/mol and −43.9 kcal/mol ≤ ∆G H -T (DH − /iAsc) ≤ −37.8 kcal/mol.What is more, since all of the Gibbs free energies of hydride transfer processes from DH 2 to Ru IV O 2+ (−114.1 kcal/mol), from DH 2 to Fl + (−78.5 kcal/mol), from DH 2 to PQ (−70.0 kcal/mol), and from DH 2 to H + (−76.0 kcal/mol) are less than 0, it can be inferred that dipines may be oxidated by heme enzyme, cytochrome P450, flavin coenzyme, coenzyme Q, and H + under suitable oxidoreductase by hydride oxidation in vivo.

Hydrogen-Atom-Donating Properties
Due to the N-H bond and C-H bond at the 1-position and 4-position of DH 2 , there are six possible elementary steps to release hydrogen atoms during the aromatization.Herein, the thermodynamic hydrogen-atom-donating abilities of DH 2 , DH − , DH 2 •+ , DH • , and DH' • , as well as the hydrogen-atom affinities of 12 common radicals (involving • , and TEMPO) [29] and coenzyme models (BNA + for NADH coenzyme, PQ for coenzyme Q, iAsc for oxidated ascorbic acid, Fl + for flavin coenzyme, and Ru IV O 2+ for heme enzyme) in acetonitrile are shown in Figure 7.   [29] and coenzyme models (BNA + for NADH coenzyme, PQ for coenzyme Q, iAsc for oxidated ascorbic acid, Fl + for flavin coenzyme, and Ru IV O 2+ for heme enzyme) in acetonitrile are shown in Figure 7. From Figure 7, the thermodynamic hydrogen-atom-donating ability scale ranges from 92.9 to 93.9 kcal/mol for DH2 releasing hydrogen atoms from N1-H, from 58.6 to 60.3 kcal/mol for DH2 releasing hydrogen atoms from C4-H, from 64.4 to 65.7 kcal/mol for DH − , from 21.3 to 34.9 kcal/mol for DH2 •+ , from 50.5 to 55.7 kcal/mol for DH • , and from 17.1 to 20.5 kcal/mol for DH' • , and the thermodynamic hydrogen-atom-donating abilities From Figure 7, the thermodynamic hydrogen-atom-donating ability scale ranges from 92.9 to 93.9 kcal/mol for DH 2 releasing hydrogen atoms from N 1 -H, from 58.6 to 60.3 kcal/mol for DH 2 releasing hydrogen atoms from C 4 -H, from 64.4 to 65.7 kcal/mol for DH − , from 21.3 to 34.9 kcal/mol for DH 2 •+ , from 50.5 to 55.7 kcal/mol for DH • , and from 17.1 to 20.5 kcal/mol for DH' • , and the thermodynamic hydrogen-atom-donating abilities increase in the order of DH 2 (N 1 -H, 92.9-93.9kcal/mol) < DH − (64.4-65.7 kcal/mol) < DH 2 (C 4 -H) (58.6-60.3kcal/mol) < DH • (50.5-55.7 kcal/mol) < DH 2 •+ (21.3-34.9kcal/mol) < DH' • (17.1-20.5 kcal/mol).According to their thermodynamic ranges, DH • , DH 2 •+ , and DH' • belong to thermodynamically strong hydrogen atom donors, DH − and DH 2 generally belong to thermodynamically medium-strong hydrogen atom donors to break C 4 -H bonds, while DH 2 belong to thermodynamically weak hydrogen atom-donors to break N 1 -H bonds.Based on the above analysis, whether DH 2 are medium-strong hydrogen atom donors or weak hydrogen atom donors depends on which hydrogen atoms DH 2 releases, N 1 -H bonds or C 4 -H bonds.Generally, DH 2 prefer to release hydrogen atoms from C 4 -H bonds (58.6-60.3kcal/mol) instead of N 1 -H bonds (92.9-93.9kcal/mol) from thermodynamics.
The above thermodynamic analyses result in the following conclusions.

Two Hydrogen Ions (Atoms) Donating Properties
Examining the structural characteristics of NADH models, they are generally N-alkyl-1,4-dihydropyridines and act as hydride carriers [27,55].Unlike common NADH models, the biggest difference between common NADH models (such as BNAH and AcrH) and DH 2 or the Hantzsch ester (YH 2 ) is that DH 2 and YH 2 have two hydrogen atoms at the N 1 -position and C 4 -position.During the aromatization process, DH 2 and YH 2 could release two hydrogen ions (atoms) or hydrogen gas from both N 1 -H and C 4 -H bonds.Therefore, the Hantzsch ester (YH 2 ) has been widely used as an excellent hydrogenation reagent to reduce unsaturated compounds by offering two hydrogen ions (atoms) [56][57][58].Furthermore, many studies also focused on the hydrogen storage properties of YH 2 and related N-heterocycles [42].In addition, some oxidoreductases, for example flavin coenzyme, coenzyme Q (Co Q), heme or nonheme iron coenzyme, and pyrroloquinoline quinone (PQQ) are also the two hydrogen ions (atoms) carriers in vivo.As a result, the thermodynamic abilities of DH 2 releasing two hydrogen ions (atoms) or H 2 , ∆G H − P (DH 2 ), ∆G 2H (DH 2 ), and ∆G H2 (DH 2 ), are vital thermodynamic parameters to evaluate the comprehensive reduction properties, and judge the oxidation feasibility of DH 2 by two hydrogen ions (atoms) carrier enzymes in vivo.
The thermodynamic abilities of DH 2 and common hydrogen carriers (HCO 2 H, H 2 , YH 2 , F420H 2 , PQH 2 , and iAscH 2 ) releasing two hydrogen ions (H − + H + ), two hydrogen atoms (2H • ), or H 2 in acetonitrile are shown in Figure 9 [27,43].It is found from Figure 9 that the Gibbs free energies of DH 2 releasing two hydrogen ions range from 83.8 to 86.6 kcal/mol, which belong to medium-strong two hydrogen ion donors.
According to the thermodynamic data, several valuable conclusions could be drawn.( [29], it is discovered that DH 2 are thermodynamically better two hydrogen ion donors than PQH 2 , and DH 2 may be oxidated by Co Q via two hydrogen ions (atoms) transfer in vivo.(4) Due to the fact that ∆G H − P (DH 2 ) values (83.8-86.6 kcal/mol) are much more negative than ∆G H − P (iAscH 2 ) (100.8 kcal/mol) [27], DH 2 are thermodynamically worse two hydrogen ions (atoms) donors, and DH 2 may be oxidated by the oxidation state of ascorbic acid (Asc) in vivo.(5) Because the ∆G H2 (DH 2 ) values (7.8-10.6 kcal/mol) are greater than ∆G H2 (H 2 ) (0.0 kcal/mol), H 2 release from DH 2 is thermodynamically unfavorable, and DH 2 are not hydrogen storage chemicals unless extra energy is being provided, such as light, electron, heat, or gas pressure.modynamic abilities of DH2 releasing two hydrogen ions (atoms) or H2, ΔGH -P(DH2), ΔG2H(DH2), and ΔGH2(DH2), are vital thermodynamic parameters to evaluate the comprehensive reduction properties, and judge the oxidation feasibility of DH2 by two hydrogen ions (atoms) carrier enzymes in vivo.
The thermodynamic abilities of DH2 and common hydrogen carriers (HCO2H, H2, YH2, F420H2, PQH2, and iAscH2) releasing two hydrogen ions (H − + H + ), two hydrogen atoms (2H • ), or H2 in acetonitrile are shown in Figure 9 [27,43].It is found from Figure 9 that the Gibbs free energies of DH2 releasing two hydrogen ions range from 83.8 to 86.6 kcal/mol, which belong to medium-strong two hydrogen ion donors.According to the thermodynamic data, several valuable conclusions could be drawn.

Application of Thermodynamic Data to Evaluate the Redox Properties of DH 2
Without doubt, the thermodynamic cards of DH 2 display the redox properties of DH 2 to reveal the possible oxidative process of DH 2 (Figure 3).For DH 2 , there are five possible initial oxidation pathways (Figure 10), including hydride oxidation (Step 1), singleelectron oxidation (Step 2), hydrogen atom oxidation from the C 4 -H bond (Step 4), hydrogen atom oxidation from the N 1 -H bond (Step 18), and proton release from the N 1 -H bond (Step 15).As hydrogen atom donors or antioxidants, DH 2 could release hydrogen atoms from the C 4 -H (Step 4) or N 1 -H (Step 18) bond.The ∆G' HD (DH 2 ) values of Step 18 (N 1 -H, 92.9-93.9kcal/mol) are ~30.0kcal/mol larger than the ∆G' HD (DH 2 ) values of Step 4 (C 4 -H, 58.6-60.3kcal/mol).Thereby, it is reasonable to deduce that DH 2 generally release hydrogen atoms from C 4 -H instead of N 1 -H during the antioxidant processes from thermodynamics.

Application of Thermodynamic Data to Evaluate the Redox Properties of DH2
Without doubt, the thermodynamic cards of DH2 display the redox properties of DH2 to reveal the possible oxidative process of DH2 (Figure 3).For DH2, there are five possible initial oxidation pathways (Figure 10), including hydride oxidation (Step 1), single-electron oxidation (Step 2), hydrogen atom oxidation from the C4-H bond (Step 4), hydrogen atom oxidation from the N1-H bond (Step 18), and proton release from the N1-H bond (Step 15).As hydrogen atom donors or antioxidants, DH2 could release hydrogen atoms from the C4-H (Step 4) or N1-H (Step 18) bond.The ΔG'HD(DH2) values of Step 18 (N1-H, 92.9-93.9kcal/mol) are ~30.0kcal/mol larger than the ΔG'HD(DH2) values of Step 4 (C4-H, 58.6α-60.3kcal/mol).Thereby, it is reasonable to deduce that DH2 generally release hydrogen atoms from C4-H instead of N1-H during the antioxidant processes from thermodynamics.

Application of Thermodynamic Data of Important Intermediates to Possible Oxidative Mechanism Judgement
The thermodynamic cards of DH 2 also could reveal the redox properties of key intermediates (Figure 3).For example, if the oxidation of DH 2 is initiated by single oxidation, the thermodynamic data of resulting DH 2 •+ are noteworthy physical parameters to diagnose possible oxidation pathways.As organic acids, DH

Application of Thermodynamic Data of Important Intermediates to Possible Oxidative Mechanism Judgement
The thermodynamic cards of DH2 also could reveal the redox properties of key intermediates (Figure 3).For example, if the oxidation of DH2 is initiated by single oxidation, the thermodynamic data of resulting DH2 •+ are noteworthy physical parameters to diagnose possible oxidation pathways.As organic acids, DH2  DH2 •+ may act as proton donors or hydrogen atom donors, which depends on the properties of substrates.If the substrates are radicals, DH2 •+ act as hydrogen atom donors.If the substrates are bases, DH2 •+ act as proton donors.Most importantly, with a combination of the oxidation potentials of DH2 and the properties of resulting DH2 •+ releasing hydrogen ions (atoms), the initial slight energy barrier (10-20 kcal/mol) of electron oxidation could be overcome by the subsequent protons transfer or hydrogen atoms transfer owning extreme thermodynamic driving forces (ΔG << 0).DH 2

Application of Thermodynamic Data of DH2 to Oxidative Mechanism Diagnosis between DH2 and iAsc
•+ may act as proton donors or hydrogen atom donors, which depends on the properties of substrates.If the substrates are radicals, DH 2 •+ act as hydrogen atom donors.If the substrates are bases, DH 2 •+ act as proton donors.Most importantly, with a combination of the oxidation potentials of DH 2 and the properties of resulting DH 2 •+ releasing hydrogen ions (atoms), the initial slight energy barrier (10-20 kcal/mol) of electron oxidation could be overcome by the subsequent protons transfer or hydrogen atoms transfer owning extreme thermodynamic driving forces (∆G << 0).

Application of Thermodynamic Data of DH 2 to Oxidative Mechanism Diagnosis between DH 2 and iAsc
Since the thermodynamic cards of dipine models' (DH 2 ) aromatization on 21 potential elementary steps have been established, they provide a precious opportunity to investigate the possible oxidative mechanism of DH 2 in vivo.Ascorbic acid (AscH 2 ) is known as an excellent electron and hydrogen atom donor [52]; however, the oxidated ascorbic acid (Asc) is actually a potential two hydrogen ions (atoms) oxidant.Because of the ortho dicarbonyl feature, Asc has the property of o-benzoquinone [59], which is confirmed by the fact that ∆G H − P (iAscH 2 ) (100.8 kcal/mol) [27] is slightly greater than ∆G H − P (PQH 2 ) (96.2 kcal/mol) [29].
Herein, the oxidative mechanism between 3H 2 and iAsc is taken as an example to represent the application of the thermodynamic cards of DH 2 to oxidative mechanism diagnosis.First of all, the thermodynamic card of iAsc accepting two hydrogen ions (atoms) on nine potential elementary steps is constructed based on our previous work (Figure 12).Subsequently, according to the thermodynamic cards of 3H 2 and iAsc, a thermodynamic analysis platform of elementary steps for the redox process between 3H 2 and iAsc without any catalyst in acetonitrile is established and shown in Figure 13.Herein, the oxidative mechanism between 3H2 and iAsc is taken as an example to represent the application of the thermodynamic cards of DH2 to oxidative mechanism diagnosis.First of all, the thermodynamic card of iAsc accepting two hydrogen ions (atoms) on nine potential elementary steps is constructed based on our previous work (Figure 12).Subsequently, according to the thermodynamic cards of 3H2 and iAsc, a thermodynamic analysis platform of elementary steps for the redox process between 3H2 and iAsc without any catalyst in acetonitrile is established and shown in Figure 13.From Figure 13, Step 13 is the concerted two hydrogen ions (atoms) transfer step from 3H2 to iAsc, 3H2 + iAsc → 3 + iAscH2, and the overall Gibbs free energy of two hydride ions (atoms) transfer from 3H2 to iAsc for Step 13 is −16.1 kcal/mol, which means the oxidation of 3H2 by iAsc is thermodynamically feasible.Moreover, as can be seen from Figure 13, six possible elementary steps are involved in the initial oxidation of 3H2 by iAsc.
Step a is the hydride transfer step from 3H2 to iAsc, 3H2 + iAsc → 3H + + iAscH − , and the Gibbs free energy of Step a is −9.Additionally, the Gibbs free energies of direct hydride transfer from 3H2 to iAsc and proton transfer from 3H + to iAscH − , Step a (hydride transfer, −9.7 kcal/mol) and Step g (proton transfer, −6.4 kcal/mol), are much less than zero, and it can be inferred that the oxidation process of 3H2 and iAsc undergoing successive hydride (Step a, −9.7 kcal/mol) Step a e −

3
Step b e − H

Step c
Step j Step i Step h Step From Figure 13, Step 13 is the concerted two hydrogen ions (atoms) transfer step from 3H 2 to iAsc, 3H 2 + iAsc → 3 + iAscH 2 , and the overall Gibbs free energy of two hydride ions (atoms) transfer from 3H 2 to iAsc for Step 13 is −16.1 kcal/mol, which means the oxidation of 3H 2 by iAsc is thermodynamically feasible.Moreover, as can be seen from Figure 13, six possible elementary steps are involved in the initial oxidation of 3H 2 by iAsc.
Step a is the hydride transfer step from 3H 2 to iAsc, 3H 2 + iAsc → 3H + + iAscH − , and the Gibbs free energy of Step a is −9.Additionally, the Gibbs free energies of direct hydride transfer from 3H 2 to iAsc and proton transfer from 3H + to iAscH − , Step a (hydride transfer, −9.7 kcal/mol) and Step g (proton transfer, −6.4 kcal/mol), are much less than zero, and it can be inferred that the oxidation process of 3H 2 and iAsc undergoing successive hydride (Step a, −9.7 kcal/mol) and proton transfer (Step g, −6.4 kcal/mol) is thermodynamically feasible.
Similarly, the oxidation of 3H 2 by iAsc processing concerted two hydrogen ions (atoms) transfer (Step f, −16.1 kcal/mol) is also thermodynamically feasible.Although the concerted two hydrogen ions (atoms) transfer (Step f ) is thermodynamically feasible, 3H 2 and iAsc molecules do not completely match each other in space structure and binding site during transition state.Therefore, the concerted two hydrogen ions (atoms) transfer (Step f ) process is reasonably ruled out.It may be that in complex environments within the body, the hydride-acceptor/amino-acid residue pairs, two radicals, or benzoquinones could oxidize dipines through a concerted two hydrogen ions (atoms) transfer step.
In summary, the oxidation of 3H 2 by iAsc may experience three possible pathways, that is, H − + H + (Step a-step g), H • + e + H + (Step c-Step j-Step g), and H • + H • (Step c-Step k).But which one pathway or pathways are the real oxidation mechanism needs further experimental verification in the lab.Beyond a doubt, the thermodynamic analyses of the redox process between 3H 2 and iAsc provide us with a unique perspective into the dipines' aromatization by quinones in vivo.

Materials and Methods
Prediction Methods.The pK a values of DH + and YH + in acetonitrile were predicted by the method developed by Luo and coworkers in 2020 at http://pka.luoszgroup.com/prediction (accessed on 9 January 2024).Prediction methods: XGBoost with RMSE = 1.79 and r 2 = 0.918 (80:20 train test split).
(3) The application of the thermodynamic data of DH 2 to oxidative mechanism diagnosis between DH 2 and iAsc: Because of the ortho dicarbonyl feature, Asc has the property of o-benzoquinone.Therefore, the oxidative mechanism between 3H 2 and iAsc is taken as an example to represent the application of the thermodynamic cards of DH 2 to oxidative mechanism diagnosis.Based on thermodynamic analyses, the oxidation of 3H 2 by iAsc may experience three possible pathways, that is, H − + H + , H • + e + H + , and H • + H • .
The thermodynamic data of dipine models' aromatization suggests that the oxidative metabolism of dipine drugs is unexpectedly complex, and may involve many active intermediates and potential elementary steps under the conditions of various oxidoreductases in vivo.Therefore, the effects and levels of electrons and hydrogen atoms, as well as hydride oxidoreductases may need sustained attention or detection during the treatment period.Without a doubt, the thermodynamic cards of dipine models could help us to understand the redox properties of DH 2 and related intermediates, and diagnose the possible mechanism and pathways of dipine metabolism in vivo.

Figure 2 .Figure 2 .
Figure 2. (a) The oxidative metabolism process of Nifedipine in vivo.(b) Some common oxidoreductases in vivo.

Figure 4 .
Figure 4.The pKa of DH + and YH + , along with the pKa of common organic acids in acetonitrile.

Figure 4 .
Figure 4.The pK a of DH + and YH + , along with the pK a of common organic acids in acetonitrile.

Molecules 2024 ,
29, x FOR PEER REVIEW 9 of 21 for coenzyme Q, iAsc for oxidated ascorbic acid, Fl + for flavin coenzyme, and Ru IV O 2+ for heme enzyme) and hydride acceptors (H + and PTZ •+ ) in acetonitrile are displayed in Figure 6.
(17.1-20.5  kcal/mol) are thermodynamically better antioxidants than iAscH − (64.1 kcal/mol).(3) Unlike hydricities, the thermodynamic hydrogen-atom-donating abilities of DH − decrease as a result of the negative charge at the N 1 -atom compared with their parents DH 2 , and the thermodynamic hydrogen-atom-donating abilities of DH − (64.4-65.7 kcal/mol) are more negative than DH 2 (58.6-60.3kcal/mol) by 4.1-7.1 kcal/mol.(4) Because the final products of DH • and DH' • releasing hydrogen atoms are the same (D and H • ), DH • → D + H • and DH' • → D + H • .When the hydrogen-atom-donating Gibbs free energies of DH • (50.5-55.7 kcal/mol) and DH' • (17.1-20.5 kcal/mol) are compared, it can also be deduced that DH • are more thermodynamically stable radicals than DH' • , and the Gibbs free energies of hydrogen atom transfer within DH • from the N 1 -position to the C 4 -position [∆G HT (DH • ) for Step 20 in Figure 3] are computed as 33.4-35.2kcal/mol, which further verify the relative stability of DH • and DH' • in solution.

Figure 9 .
Figure 9. Thermodynamic abilities of DH2 and common hydrogen carriers releasing two hydrogen ions (H − + H + ) in the red brackets, releasing two hydrogen atoms (2H • ) in the purple brackets, and releasing H2 in the blue brackets in acetonitrile (kcal/mol).

Figure 11 .
Figure 11.Thermodynamic analysis of possible oxidative process for intermediate DH 2 •+ .For DH 2 •+ , there are three possible pathways to release hydrogen ions or atoms in all (Figure 11), including proton release from C 4 -H bonds (Step 6), proton release from N 1 -H bonds (Step 21), and hydrogen atom release from C 4 -H bonds (Step 5).According to their thermodynamic results (Step 6, Step 21, and Step 5), DH 2 •+ belong to strong proton donors for C 4 -H bond breaking (Step 6: −9.3-−13.2kcal/mol), medium-strong proton donors for N 1 -H bond breaking (Step 21: 22.0-24.1 kcal/mol), and strong hydrogen atom donors for C 4 -H bond breaking (Step 5: 21.3-24.9kcal/mol).DH 2•+ may act as proton donors or hydrogen atom donors, which depends on the properties of substrates.If the substrates are radicals, DH 2•+ act as hydrogen atom donors.If the substrates are bases, DH 2•+ act as proton donors.Most importantly, with a combination of the oxidation potentials of DH 2 and the properties of resulting DH 2•+ releasing hydrogen ions (atoms), the initial slight energy barrier (10-20 kcal/mol) of electron oxidation could be overcome by the subsequent protons transfer or hydrogen atoms transfer owning extreme thermodynamic driving forces (∆G << 0).

Figure 12 .
Figure 12.Thermodynamic card of iAsc accepting two hydrogen ions (atoms) on nine potential elementary steps.

Figure 12 . 21 Figure 13 .
Figure 12.Thermodynamic card of iAsc accepting two hydrogen ions (atoms) on nine potential elementary steps.024, 29, x FOR PEER REVIEW 17 of 21 7 kcal/mol.Step b is the electron transfer step from 3H 2 to iAsc, 3H 2 + iAsc → 3H •+ + iAsc •− , and the Gibbs free energy of Step b is 31.4kcal/mol.Steps c and d are the hydrogen atom transfer step from C 4 -H and N 1 -H bonds in 3H 2 to iAsc, respectively, 3H 2 + iAsc → 3H • + iAscH • and 3H 2 + iAsc → 3H' • + iAscH • , and the Gibbs free energy of Steps c and d are 0.0 and 34.2 kcal/mol.Step e is the proton transfer step from 3H 2 to iAsc, 3H 2 + iAsc → 3H − + iAscH + , and the Gibbs free energy of Step e is 46.5 kcal/mol.Finally, Step f is the concerted two hydrogen ions (atoms) transfer step from 3H 2 to iAsc, and the Gibbs free energy of Step f is −16.1 kcal/mol.According to the thermodynamic potentials of Steps a-f, the Gibbs free energies of Step b (31.4 kcal/mol), Step d (34.2 kcal/mol), and Step e (46.8 kcal/mol) are greater than 30 kcal/mol, which implies that the oxidation of 3H 2 could not occur through electron transfer (Step b), hydrogen transfer from the N 1 -H bond (Step d), and proton transfer (Step e).Since the Gibbs free energies of Step a (−9.7 kcal/mol), Step c (0.0 kcal/mol), and Step f (−16.1 kcal/mol) are less than or equal to zero, the oxidation of 3H 2 may experience an initial hydride (Step a), hydrogen atom from C 4 -H (Step c), and concerted two hydrogen ions (atoms) transfer step (Step f ).Further investigating the thermodynamics of possible hydrogen atom oxidation processes, there are two different pathways for the oxidation of 3H 2 by iAsc overall.The first is the H • + e + H + pathway (Step c-Step j-Step g), and the second is the H • + H • pathway (Step c-Step k).Due to the fact that all of the Gibbs free energies of the above five elementary steps are less than or equal to zero (−16.1-0.0 kcal/mol), it can be assumed that these two hydrogen-atom-initiated pathways, Step c-Step j-Step g and Step c-Step k, are thermodynamically feasible. 2 •+ ) for Step 5, ∆G HD (DH − ) for Step 10, ∆G HD (DH' • ) for Step 11, ∆G HD (DH • ) for Step 17, and ∆G' HD (DH 2 ) for Step 18, respectively.Based on Hess' law 2 2 , and (17) in Table 1, respectively, by constructing corresponding thermodynamic cycles, cycle Step 4-Step 2-Step 6 for ∆G PD (DH 2 •+ ), cycle Step 8-Step 12-Step 10 for ∆G PD (DH' • ), cycle Step 14-Step 15-Step 7 for ∆G PD (DH 2 ), and cycle Step 6-Step 20-Step 21 for ∆G' PD (DH 2 •+ ).

41.2 kcal/mol 31.4 kcal/mol 34.2 kcal/mol H Step k −16.1 kcal/mol H
Thermodynamic analysis platform of elementary steps for the redox process between 3H 2 and iAsc without any catalyst in acetonitrile.